BACKGROUND: Although esculetin, a coumarin compound, is known to induce apoptosis in human cancer cells, the effects and molecular mechanisms on the apoptosis in human malignant melanoma (HMM) cells are not well understood yet. In this study, we investigated the anti-proliferative effects of esculetin on the G361 HMM cells. METHODS: We analyzed the anti-proliferative effects and molecular mechanisms of esculetin on G361 cells by a 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, 4\',6-diamidino-2-phenylindole staining and Western blotting. RESULTS: Esculetin exhibited significant anti-proliferative effects on the HMM cells in a dose-dependent manner. Interestingly, we found that esculetin induced nuclear shrinkage and fragmentation, typical apoptosis markers, by suppression of Sp1 transcription factor (Sp1). Notably, esculetin modulated Sp1 downstream target genes including p27, p21 and cyclin D1, resulted in activation of apoptosis signaling molecules such as caspase-3 and PARP in G361 HMM cells. CONCLUSIONS: Our results clearly demonstrated that esculetin induced apoptosis in the HMM cells by downregulating Sp1 protein levels. Thus, we suggest that esculetin may be a potential anti-proliferative agent that induces apoptotic cell death in G361 HMM cells.
Apoptosis ; Blotting, Western ; Caspase 3 ; Cell Death ; Cyclin D1 ; Humans* ; Melanoma* ; Sp1 Transcription Factor

Xenotransplantation, the transplantation of cells, tissues or organs between individuals of different species, would resolve the current shortage of organs, but rejection remains the major hurdle to successful xenotransplantation. In the present study, we analyzed mixed lymphocyte reactions (MLRs) and used 51Cr release assays in order to identify the proliferation and expansion of mouse CD8+ cytotoxic T lymphocyte cells against PK15, PK15/pIL-18 or PK15/mIL-18 cells. In addition, we identified T cell populations in mouse splenocytes and lymph node cells using two-color flow cytometry. It was found that the CD8+T cells of xenograft recipients proliferated extensively and that the survival rates of populations of PK15/mIL-18 or PK15/pIL-18 cells were higher than untransfected controls. Moreover, CD3+T cells were increased in mice injected with PK15 cells or PK15/pIL-18 cells but PK15/pIL-18 cell numbers were lower in lymph nodes than untransfected controls. CD8+T cells numbers were reduced in the lymph nodes of PK15/pIL-18 injected mice. These results suggest that porcine IL-18 regulates anti-pig cellular rejection in C57BL/6 mice.
Transplantation, Heterologous ; Transplantation ; Transgenes/immunology/physiology ; Transfection ; Tissue Distribution ; T-Lymphocytes/metabolism ; Swine ; RNA, Messenger/metabolism ; Phenotype ; Mice, Inbred C57BL ; Mice ; Lymphocyte Culture Test, Mixed ; Lymphocyte Activation ; Kidney/cytology/*immunology ; Interleukin-18/*genetics/metabolism/physiology ; Immunosuppression/*methods ; Graft Rejection/immunology/*prevention & control ; Genetic Vectors/chemical synthesis ; Female ; Epithelial Cells/*drug effects/*transplantation ; Cytokines/metabolism ; Cells, Cultured ; Animals

Stromal cell-derived factor 1 (SDF-1/CXCL12) is a multifunctional cytokine implicated in normal hematopoiesis. We previously reported that SDF-1alpha enhanced the survival of hematopoietic stem and progenitor cells in synergy with other cytokine such as GM-CSF, steel factor, or thrombopoietin. As adult stem cells are very rare, many investigators are trying to expand hematopoietic stem/progenitor cells in vitro. In this study, we constructed an adenoviral vector and produced high titer of recombinant adenoviruses directing robust expression of SDF-1alpha determined by ELISA. We also produced control empty adenoviruses and recombinant LacZ adenoviruses. In order to check the feasibility of SDF-1alpha in ex vivo expansion system, we compared HUVEC cells tranduced by a SDF-1alpha recombinant virus with HUVEC cells transduced by a LacZ recombinant virus in supporting activity of hematopoietic cells, and found that expression of SDF-1alpha in HUVEC cells increased viable blood cell population obtained from the same number of CD34+ cells. The SDF-1alpha recombinant adenovirus seems to be useful for future application in hematopoiesis studies.
Adenoviridae* ; Adult Stem Cells ; Blood Cells ; Chemokine CXCL12* ; Enzyme-Linked Immunosorbent Assay ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hematopoiesis ; Hematopoietic Stem Cells* ; Human Umbilical Vein Endothelial Cells ; Humans ; Research Personnel ; Stem Cell Factor ; Stem Cells ; Thrombopoietin

BACKGROUND: Biomedical products such as viral vaccines can be contaminated with hazardous viruses during manufacturing processes and storage, thus causing harmful side effects. To assure the safety of biomedical products, highly effective and sensitive methods should be available to detect contaminating viruses. In this study, we performed recovery tests to determine the limit of detection of HIV-1. METHODS: An HIV-1 plasmid preparation was serially diluted and spiked into various culture media (DMEM, RPMI-1640, IMDM, GICM, and SDM) containing 10% fetal bovine serum (FBS). The HIV-1 plasmid was detected by PCR alone or a combination of PCR and ELISA (PCR/ELISA). RESULTS: When spiked into DMEM, RPMI, and IMDM, less than 4x10(-2) ng of HIV-1 plasmid was not detectable as HIV-1 PCR products in agarose gel. Intra- and inter-assays (n=6) showed that the PCR-ELISA system could detect PCR products diluted as much as 1, 875 times from HIV-1 plasmid serially spiked in various media. CONCLUSIONS: The PCR/ELISA system can be useful for the detection of trace amounts of hazardous viruses which may be present as contaminants in biological products.
Biological Products ; Culture Media ; Enzyme-Linked Immunosorbent Assay ; HIV-1* ; Limit of Detection ; Plasmids ; Polymerase Chain Reaction ; Sepharose ; Viral Vaccines

In the early host defense system, effector function of natural killer (NK) cells results in natural killing against target cells such as microbe-infected, malignant, and certain allogenic cells without prior stimulation. NK cell cytotoxicity is selectively regulated by homeostatic prevalence between a repertoire of both activating and inhibitory receptors, and the discrimination of untransformed cells is achieved by recognition of major histocompatibility complex (MHC) class I alleles through inhibitory signals. Although it is well known that the bipotential T/NK progenitors are derived from the common precusor, functional mechanisms in terms of the development of NK cells remain to be further investigated. NK cells are mainly involved in innate immunity, but recent studies have been reported that they also play a critical role in adaptive immune responses through interaction with dendritic cells (DC). This interaction will provide effector functions and development of NK cells, and elucidation of its precise mechanism may lead to therapeutic strategies for effective treatment of several immune diseases.
Adaptive Immunity* ; Alleles ; Dendritic Cells ; Discrimination (Psychology) ; Homicide ; Immune System Diseases ; Immunity, Innate ; Killer Cells, Natural* ; Major Histocompatibility Complex ; Prevalence

Thymus is a lymphoid organ forming T-cells from hematogenous stem cells. There is no report on the germinal centers in the thymus except the Myasthenia gravis in human. In this study, new germinal centers were experimentally induced from 6 days after 5-FU treatment in the Sprague-Dawley rats. With germinal center formation, proteins of molecular weight 123 kDa on 6 days after 5-FU treatment, and 162 kDa and 205 kDa on 9 days after 5-FU treatment were increased in 5-FU treated group. These proteins revealed alpha 1 -and beta 1 -integrins on integrin Western Blot. In this experiment, it was cosidered that alpha 1-integrins and beta 1 -integrins were the key proteins for proliferation of B cells within the newly formed thymic germinal centers and the massive apoptotic disposal of B-cells from the germinal centers, and the new formation of T cells within the cortex.
Animals ; B-Lymphocytes ; Blotting, Western ; Fluorouracil* ; Germinal Center* ; Humans ; Integrins* ; Molecular Weight ; Myasthenia Gravis ; Rats* ; Rats, Sprague-Dawley ; Stem Cells ; T-Lymphocytes ; Thymus Gland*

Although cadmium is a well known heavy metal which has an influence testis and brings about male infertility, the mechanism of action in the testis is still fully unknown. In these experiment, cadmium chloride 4 mg/kg of body weight administered intraperitoneally to the rat (Sprague-Dawley) and sacrificed after 1 week, and morphological changes were observed by LM and TEM. In addition, electrophoresis, immunoprecipitation and Western blotting and N-terminal analysis performed to reveal the protein changes. 1. Major findings under light microscope were hemorrhagic necrosis and death of all the spermatogenic cells and supporting cells within the seminiferous tubules, and decreased volume of ECM, many apoptotic bodies, and death of interstitial cells and fibroblasts within interstitium. 2. The EM findings were disruption of nuclear membrane and disappearance of cell organelles of spermatogenic cells and supporting cells within seminiferous tubules, and decreased filopodia, increased inclusion bodies, vacuolation and apoptotic changes of the interstitial cells and fibroblastic cells, many short electron-dense collagen fibers in the extracellular matrix of interstitium. 3. Two proteins of molecular weight 42 kDa and 21 kDa which disappeared after cadmium treatment were rat collagen type I alpha 2. According to the above results, it is considered that cadmium degrades the collagen of the wall of small blood vessels within seminiferous tubules and interstitium and disrupts vascular walls, which results hemorrhagic necrosis, death of all the spermatogenic cells, and the death of interstitial cells and fibroblastic cells.
Animals ; Blood Vessels ; Blotting, Western ; Body Weight ; Cadmium Chloride* ; Cadmium* ; Collagen ; Collagen Type I ; Electrophoresis ; Extracellular Matrix ; Fibroblasts ; Immunoprecipitation ; Inclusion Bodies ; Infertility, Male ; Male ; Molecular Weight ; Necrosis ; Nuclear Envelope ; Organelles ; Pseudopodia ; Rats* ; Seminiferous Tubules ; Testis*

BACKGROUND AND OBJECTIVES: Dual-chamber pacing is commonly used as a pacing mode maintaining at-rioventricular synchrony. However, traditional technique for atrial J lead implantation is relatively time-consuming and not easy to get optimal sites for both atrial and ventricular leads, especially for less-experienced operators. We developed a new "push technique" for atrial J lead implantation. MATERIALS AND METHOD: This study included sixty-two consecutive patients (26 males 36 females, 55+/-10 years, 56 patients received DDD and 6 received AAI pacemakers) from Jan. 1992 through Dec. 1996. Atrial J leads were implanted using a "push technique", that is, simply advancing a straightened lead while holding the stylet in the pacing lead at the junction of the superior vena cava and the right atrium. We evaluated the early and long-term result of atrial J lead implantation by the "push technique". RESULTS: 1) Atrial leads were successfully inserted by the first or second trial of the "push technique" in the 62 patients (100%). The sensed P wave amplitude was 3.1+/-1.0 mV, pacing threshold 0.6+/-0.2 V at the pulse width of 0.5 ms, impedance 547.4+/-118.5 ohms at 5 V. 2) During follow-up of 28.1+/-15.7 months, significant changes in the pacing parameters and the dislodgement of atrial leads were not seen. CONCLUSION: The early and long-term result of atrial J lead implantation by the "push technique" was excellent. This "push technique" can be used as an easy alternative technique for atrial J lead implantation.
Dichlorodiphenyldichloroethane ; Electric Impedance ; Female ; Follow-Up Studies ; Heart Atria ; Humans ; Male ; Vena Cava, Superior

Effusive constrictive pericarditis after open heart surgery is a rare complication occuring in 0.2% to 0.3%. Presenting symptoms after surgery are associated with right heart failure and an elevated jugular venous pressure is most common abnormal physical sign. Predisposing factors include hemorrhage, perioperative pericardial injury or inflammation, presence of postpericardiotomy syndrome and open pericardium. Early diagnosis is important because(1) if it is unrecognized, the patient may deteriorate clinically, and(2) if surgery is delayed, the patient may have an increased risk of operative death. Hereby we report a case of effusive constrictive pericarditis after ventricular septal defect repair, in which constriction physiology was suggested by Doppler echocardiography after pericardiostomy.
Causality ; Constriction ; Early Diagnosis ; Echocardiography, Doppler ; Heart Failure ; Heart Septal Defects, Ventricular* ; Hemorrhage ; Humans ; Inflammation ; Pericardial Window Techniques ; Pericarditis, Constrictive* ; Pericardium ; Physiology ; Postpericardiotomy Syndrome ; Thoracic Surgery ; Venous Pressure

BACKGROUND: Acute or subacute stent thrombosis, bleeding complications and restenosis remain major clinical concerns in coronary stenting despite high pressure inflation and intravascular ultrasound guidance. A new strategy of local heparin delivery may maintain sustained local concentration and limit systemic complications. To observe the feasibility and efficacy of local heparin delivery in stenting, local heparin deliveries were performed in stented patients. METHOD: Heparin was delivered(5,000 Units, 1.0ml/min over 10 min) using the Dispatch Catheter, after predilation of target lesons in 10 patients(4 unstable angina, 6 acute myocardial infarction, mean age 52+/-7 yr) in the left anterior descending artery without systemic heparin loading. After local heparin delivery. Palmaz-Schatz stents were placed using standard methods. APTT and CK were checked at 1hr, 3hrs and 24 hrs after local heparin delivery and stenting. Follow-up coronary angiograms were done at 48 hrs and 6 months after stenting. RESULTS: All patients had no ischemic symptoms or ECG changes during and after local heparin delivery. All APTT and CK values were unchanged at 3 hrs and 24 hrs after local heparin delivery and stenting. Follow-up quantitative coronary angiograms at 48 hrs and 6 months showed all stents patent, with TIMI III distal flow, and without intra-stent thrombus(%diameter stenosis : 79.4+/-4.2% before predilation, 32.9+/-7.7% after predilation, 32.4+/-13.1% after local delivery, 14.2+/-2.3% immediately after stenting, 13.9+/-2.5% at 48 hrs and 21.7+/-8.8% at 6 months after stenting). CONCLUSION: Intracoronary stenting may be performed safely and effectively without systemic heparin therapy by using local heparin prior to stent implantation. Long-term stent patency and lack of coronary events appear favorable.
Angina, Unstable ; Arteries ; Catheters ; Constriction, Pathologic ; Electrocardiography ; Follow-Up Studies ; Hemorrhage ; Heparin* ; Humans ; Inflation, Economic ; Myocardial Infarction ; Stents* ; Thrombosis ; Ultrasonography